Breaking a Dogma: High-Throughput Live-Cell Imaging in Real-Time with Hoechst 33342

Automated high-throughput live cell imaging (LCI) enables investigation of substance effects on cells in vitro. Usually, cell number is analyzed by phase-contrast imaging, which is reliable only for a few cell types. Therefore, an accurate cell counting method, such as staining the nuclei with Hoechst 33342 before LCI, will be desirable. However, since the mid-1980s, the dogma exists that Hoechst can only be used for endpoint analyses because of its cytotoxic properties and the potentially phototoxic effects of the excitation light. Since microscopic camera sensitivity has significantly improved, this study investigates whether this dogma is still justified. Therefore, exposure parameters are optimized using a 4× objective, and the minimum required Hoechst concentration is evaluated, allowing LCI at 30-min intervals over 5 days. Remarkably, a Hoechst concentration of only 57 × 10^-9 m significantly inhibits proliferation and thus impairs cell viability. However, Hoechst concentrations between 7 × 10^-9  and 28 × 10^-9 m can be determined, which are neither cytotoxic nor impacting cell viability, proliferation, or signaling pathways. The method can be adapted to regular inverted fluorescence microscopes and allows, for example, to determine the cytotoxicity of a substance or the transduction efficiency, with the advantage that the analysis can be repeated at any desired time point.